The scientific goal of this proposal is to test the roles of estrogen and interleukin (IL)-1?potent autacoids known to exacerbate inflammation, on a particularly severe cause of pelvic pain associated with deep infiltrating endometriosis (DIE). Endometriosis affects as many as 10% of reproductive age women and accounts for >$22 billion annually in direct and indirect medical costs here in the USA. DIE, wherein ectopic implants penetrate >5 mm into the subperitoneal space, represents the most painful manifestation of the disease. It is reported that ~95% of women with this type of endometriosis experience moderate to severe pain, commonly presenting with symptoms of dysmenorrhea (painful periods), dyschezia (painful bowel movements) and dyspareunia (painful intercourse). DIE lesions are imbued with dense nerve and vascular networks that we postulate arise via an integrated developmental program we have coined neuroangiogenesis. We have identified one multifunctional protein, brain-derived neurotrophic factor (BDNF), that is a candidate neuroangiogenesis mediator in DIE, which we will investigate in detail under the aegis of this exploratory R21 grant. BDNF is mitogenic for both neurons and endothelial cells and it is overexpressed in ectopic and eutopic endometrium of women with endometriosis compared to controls without the disease. Moreover, both mRNA and protein of its cognate receptor, tyrosine kinase B (TrkB), are upregulated in endometriosis cases. Thus, BDNF exhibits the expression pattern and biochemical characteristics expected of a neuroangiogenesis mediator. We postulate that this protein contributes to DIE lesion growth, vascularization and pain. Preoperative pain and quality of life instruments will be collected for comparison with the biochemcial data. We will validate the cellular distribution of BDNF mRNA and protein in DIE biopsies using in situ molecular histochemical assays. Normal endometrium from women without endometriosis or pain will serve as a control. Isoforms of BDNF will be evaluated by Western blotting. The aforementioned descriptive studies will be accompanied by mechanistic experiments, using cell culture models that we developed and characterized. Control and endometriosis cell cultures will be used to evaluate the effects of estrogens and IL- 1?n BDNF mRNA and protein regulation in vitro, including the intracellular processing of pro-BDNF to its mature, bioactive form. Our preliminary data indicate that estrogens and IL-1?timulate BDNF production. Dose-response and time-course experiments, along with pharmacological inhibitors and siRNA knock down of ER?ER?GPER or IL-1 type I receptor (using scrambled siRNA as a control) will identify mechanism-specific drug targets for neuroangiogenesis, so that effective therapeutics can be rationally designed for endometriosis- associated pain in the future. Two novel estrogen receptor antagonists, synthesized and provided by our collaborators, also will be evaluated. At the completion of the R21 period, an R01 application will be prepared to address the relevant biological activities of BDNF-induced nerve and capillary proliferation and migration and their correlation with pelvic pain symptoms.